pls-08-01074 June 23, 2017 Time: 16:56 # 1
ORIGINAL RESEARCH
published: 26 June 2017
doi: 10.3389/ pls.2017.01074
Edi ed by:
José M. G ünzweig,
Heb ew Uni e si y o Je usalem, Is ael
Re iewed by:
Ch is ophe Thaye Cole,
Uni e si y o Wisconsin–Madison,
Uni ed S a es
Tanya Handa,
Uni e si é du Québec à Mon éal,
Canada
*Co espondence:
Juha Mikola
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Func ional Plan Ecology,
a sec ion o he jou nal
F on ie s in Plan Science
Recei ed: 03 Janua y 2017
Accep ed: 06 June 2017
Published: 26 June 2017
Ci a ion:
Paaso U, Keski-Saa i S, Keinänen M,
Ka inen H, Sil e T, Rousi M and
Mikola J (2017) In apopula ion
Geno ypic Va ia ion o Folia
Seconda y Chemis y du ing Lea
Senescence and Li e Decomposi ion
in Sil e Bi ch (Be ula pendula).
F on . Plan Sci. 8:1074.
doi: 10.3389/ pls.2017.01074
In apopula ion Geno ypic Va ia ion
o Folia Seconda y Chemis y du ing
Lea Senescence and Li e
Decomposi ion in Sil e Bi ch
(Be ula pendula)
Ulla Paaso1, Sa i a Keski-Saa i2, Ma kku Keinänen2, Heini Ka inen1, Ta ja Sil e 1,
Ma i Rousi3and Juha Mikola1*
1Depa men o En i onmen al Sciences, Uni e si y o Helsinki, Lah i, Finland, 2Depa men o En i onmen al and Biological
Sciences, Uni e si y o Eas e n Finland, Joensuu, Finland, 3Na u al Resou ces Ins i u e Finland (Luke), Helsinki, Finland
Abundan seconda y me aboli es, such as condensed annins, and hei in e popula ion
geno ypic a ia ion can emain h ough plan lea senescence and a ec li e
decomposi ion. Whe he he in apopula ion geno ypic a ia ion o a mo e di e se
asso men o seconda y me aboli es equally pe sis s h ough lea senescence and
li e decomposi ion is no well unde s ood. We analyzed concen a ions o in acellula
phenolics, epicu icula la onoid aglycones, epicu icula i e penoids, condensed
annins, and lignin in g een lea es, senescen lea es and pa ly decomposed li e o
sil e bi ch, Be ula pendula. B oad-sense he i abili y (H2) and coe icien o geno ypic
a ia ion (CVG) we e es ima ed o me aboli es in senescen lea es and li e using 19
geno ypes selec ed om a B. pendula popula ion in sou he n Finland. We ound ha
mos o he seconda y me aboli es emained h ough senescence and decomposi ion
and ha hei pe sis ence was ela ed o hei chemical p ope ies. In apopula ion H2
and CVG o in acellula phenolics, epicu icula la onoid aglycones and condensed
annins we e high and ema kably, inc eased om senescen lea es o decomposed
li e . The ank o geno ypes in me aboli e concen a ions was pe sis en h ough li e
decomposi ion. Lignin was an excep ion, howe e , wi h a diminishing geno ypic a ia ion
du ing decomposi ion, and he concen a ions o lignin and condensed annins had a
nega i e geno ypic co ela ion in he senescen lea es. Ou esul s show ha seconda y
me aboli es and hei in apopula ion geno ypic a ia ion can o he mos pa emain
h ough lea senescence and ea ly decomposi ion, which is a p e equisi e o ini ial li e
quali y o p edic a ia ion in li e decomposi ion a es. Pe sis en geno ypic a ia ion
also opens an a enue o selec ion o impac li e decomposi ion in B. pendula
popula ions h ough ac ing on hei g een oliage seconda y chemis y. The nega i e
geno ypic co ela ions and diminishing he i abili y o lignin concen a ions may, howe e ,
coun e ac his p ocess.
Keywo ds: condensed annins, geno ypic a ia ion, he i abili y, lea li e decomposi ion, lignin, phenolic
compounds, seconda y me aboli es, i e penoids
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Paaso e al. Seconda y Chemis y o Bi ch Li e
INTRODUCTION
Plan s p oduce an abundance o di e se seconda y me aboli es
such as phenolics and e penoids. These compounds we e
hough o be was e p oduc s un il F aenkel (1959) ecognized
ha hey ha e an impo an ole in he bi o e de ense. Since
his ea ly disco e y, unde s anding o he ole o seconda y
me aboli es in plan ecology has g ea ly expanded (Theis and
Le dau, 2003). Besides ac ing as an i-he bi o y agen s (Haukioja,
2003;Ma emyano e al., 2015), seconda y me aboli es can
de end plan s agains mic obial a ack (Dixon, 2001;Ped as
e al., 2003), egula e in e ac ions wi h bene icial mic obes such
as myco hizal ungi (Akiyama e al., 2005) and p o ec plan s
agains UV adia ion (Tegelbe g e al., 2001;Keski-Saa i e al.,
2005). Seconda y me aboli es can also emain in lea li e , and
by a ec ing soil mic obial ac i i y, li e decomposi ion, and
nu ien cycling ha e e ec s on ecosys em unc ioning (No hup
e al., 1998;Hä enschwile and Vi ousek, 2000;Schwei ze
e al., 2004;Ko ilainen e al., 2009). While hese ‘a e li e’ e ec s
a e widely ecognized, unde s anding o me aboli e dynamics
in plan li e is o en based on one dominan g oup o
compounds such as condensed annins (e.g., Schwei ze e al.,
2004, 2008b) and s udies on he pe sis ence o a wide ange
o indi idual compounds (e.g., Galle and Leb e on, 1995) ha e
emained sca ce. Focusing mo e on he di e si y o less abundan
me aboli es is encou aged by a ecen hypo hesis ha p oduc ion
o low-abundance, low-molecula weigh seconda y compounds
may se e as a mechanism o ees in opical o es s o en o ce
ene gy s a a ion o soil decompose s and conse e he nu ien s
in li e , whe e hey a e accessible by plan -associa ed myco hizal
ungi (Hä enschwile e al., 2011).
The dynamics o seconda y me aboli es du ing lea senescence
and li e decomposi ion can be linked o hei chemical s uc u e
(Galle and Leb e on, 1995), bu only a ely ha e such s udies
been ca ied ou in su icien de ail o sc u inized in e ms o
he chemical p ope ies o he compounds. Small di e ences in
he chemical s uc u e o he me aboli es may s ongly a ec
he a es o chemical p ocesses du ing decomposi ion. Fo
example, in he senescen lea es and shed lea li e , dis up ion
o cellula memb anes eleases phenolics om acuoles in o
cy osol, whe e hey a e exposed o he enzyma ic ac i i y o
polyphenol oxidases (PPOs). Compounds wi h ca echol moie y
(o ho-diphenol) a e p e e en ial subs a es o PPOs (Rawel
and Rohn, 2010), and a e he e o e oxidized mo e apidly
han compounds ha lack icinal hyd oxyl g oups in hei
phenolic ing s uc u e. Such di e ences may de e mine he
a e o compounds du ing decomposi ion as was demons a ed
o 14C-labeled pa a- and o ho-hyd oxybenzoic acids in aiga
o es soils (Sugai and Schimel, 1993). Phenolic compounds a y
widely in hei s uc u e and some o hem, such as la onoid
glycosides and phenolic acids, dec ease in concen a ion al eady
du ing lea senescence, while o he s, pa icula ly polyme s (such
as lignin and condensed annins), wi hs and decomposi ion
(Galle and Leb e on, 1995). S uc u al di e ences may also
a ec o he chemical p ope ies ele an in decomposi ion, such
as he hyd ophobici y and oxici y o he decomposing mic o-
o ganisms.
Th ee al e na i e hypo heses we e ecen ly o mula ed
ega ding he ac o s ha con ol li e chemis y du ing
decomposi ion (Wickings e al., 2012). The chemical con e gence
hypo hesis s a es ha plan li e s s a esembling each o he
o e he cou se o decomposi ion, while he ini ial li e quali y
hypo hesis p oposes ha he ini ial chemis y can be used
o simula e he quali y h oughou he decomposi ion. The
decompose con ol hypo hesis unde lines he impo ance
o dis inc decompose communi ies in luencing he li e
chemis y du ing decomposi ion. Wickings e al. (2012) ound
ha he chemis y o di e en li e ypes di e ged a he han
con e ged, bu hei esul s also show ha he h ee hypo heses
a e no mu ually exclusi e: he decompose communi ies ha e
a key ole in egula ing changes in li e chemis y al hough he
e ec s depend s ongly on he ini ial li e quali y. Recen s udies
ha e also ound suppo o he chemical con e gence hypo hesis
(Wallens ein e al., 2013;Pa sons e al., 2014), bu conclude ha
his may be an o e simpli ica ion.
La ge in e speci ic a ia ion in he composi ion and quan i y
o seconda y me aboli es is known o lead o p o ound di e ences
in soil o ganic ma e accumula ion and nu ien cycling
in e es ial ecosys ems (Wa dle e al., 1997). In aspeci ic
geno ypic a ia ion in seconda y me aboli es can be equally
subs an ial as shown o ee species in Be ula (Keinänen e al.,
1999;Lai inen e al., 2000, 2005), Populus (Schwei ze e al.,
2008b), Alnus (Lece and Chau e , 2008), and Salix (Heiska e al.,
2007). Gene ic a ia ion is a p e equisi e o na u al selec ion
and e olu ion, bu ecen e idence sugges s ha i can also shape
local communi ies and con ol ecosys em unc ioning, especially
when ound in a dominan plan species (Whi ham e al., 2008;
Genung e al., 2011;Pas o , 2017). T ee geno ypes a e known
o di e in he composi ion o ungal and insec communi ies
in hei canopies (Ba bou e al., 2009) and h ough lea li e
all, o a ec he composi ion and unc ioning o soil mic obial
communi ies (Schwei ze e al., 2008a;Mad i ch and Lind o h,
2011). Di e ences in li e quali y wi hin a popula ion can also
lead o di e ences in ca bon and ni ogen luxes (Mad i ch and
Hun e , 2005). In many o hese e ec s, seconda y me aboli es
play a c ucial ole (Schwei ze e al., 2008a;Ba bou e al., 2009;
Mad i ch and Lind o h, 2011), sugges ing ha hese compounds
may be pa icula ly help ul in e ealing how na u al selec ion,
ac ing on he gene ic s uc u e o a dominan plan popula ion,
can d i e communi y composi ion and ecosys em unc ioning.
In his s udy, we ocus on he a e o olia seconda y
me aboli es and he pe sis ence o hei geno ypic a ia ion
h ough lea senescence and li e decomposi ion in a Be ula
pendula Ro h popula ion. Be ula pendula is a common, as -
g owing deciduous ee in he no he n, and eas e n Eu ope
(A kinson, 1992), whe e i o en domina es ea ly bo eal o es
succession. Due o i s ecological and economic impo ance in
he no he n a eas, he in a- and in e popula ion geno ypic
a ia ion o B. pendula ai s ha e been a subjec o in ensi e
esea ch. The s udies ha e co e ed ee g ow h (P i inen
e al., 2003;Sil e e al., 2009;Mikola e al., 2014) and
physiology (Sil e e al., 2008;Possen e al., 2014) as well
as he bi o e suscep ibili y (Rousi e al., 1991, 1997;Pusenius
e al., 2002;Sinkkonen e al., 2012) and decomposi ion o
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Paaso e al. Seconda y Chemis y o Bi ch Li e
lea li e (Sil e e al., 2007, 2015). Folia seconda y me aboli es
(Keinänen and Julkunen-Tii o, 1998;Lai inen e al., 2000), hei
geno ypic a ia ion and ole in he bi o e and s ess esponses
(Mu ikainen e al., 2000;Yamaji e al., 2003) a e equally well
known o B. pendula. Fo example, he la onoid aglycones and
i e penoids ound on B. pendula lea su ace can impai he
g ow h and su i al o Lyman ia dispa la ae (Ma emyano
e al., 2015). The abili y o seconda y me aboli es o explain he
link be ween he bi o e esis ance and li e decomposi ion a e
in B. pendula has also been es ed, wi h no ob ious ole ound
(Sil e e al., 2015), bu he basic knowledge o al e a ions in
he seconda y me aboli e p o iles and hei geno ypic a ia ion
in lea senescence and li e decomposi ion is lacking. Ou s udy
was designed o ill his gap o knowledge. We hypo hesized
ha seconda y me aboli es, anging om he ample condensed
annins and lignin o o he phenolics and i e penoids o lowe
concen a ions (1) emain h ough lea senescence, (2) exhibi
signi ican geno ypic a ia ion in he senescen B. pendula lea es,
and (3) emain and p ese e hei geno ypic a ia ion h ough
he ea ly phase o li e decomposi ion. These hypo heses, i
suppo ed, would mani es he pe sis ence o geno ypic a ia ion
o olia chemis y h ough senescence and decomposi ion: a
p e equisi e o he ini ial li e quali y o p edic and he
selec ion ac ing on olia chemis y o a ec li e decomposi ion.
MATERIALS AND METHODS
Field Si es, Plan Ma e ial, and Lea
Sampling
The lea ma e ial was collec ed om he Kuikannii y
expe imen al si e, es ablished in 1999 on an abandoned
ag icul u al ield in Punkaha ju, sou heas Finland (61◦470N,
29◦210E). The ees ha g ow in Kuikannii y consis o he
mic op opaga ed p ogeny o 30 B. pendula ees (Lai inen e al.,
2005), selec ed om a nea by 0.9-ha o es s and (Lai inen e al.,
2000). The sou ce s and was na u ally egene a ed a e 1979
logging and he selec ed mo he ees g ow in six g oups, loca ed
10–60 m apa . The Kuikannii y si e consis s o six eplica e
blocks, each including wo ees o each o he 30 geno ypes.
Nine een o hese geno ypes we e used in his s udy (excep
ha g een lea measu emen s we e es ic ed o eigh geno ypes
a e quali y assessmen o he analyses, whe e accumula ion
o polyme ic subs ances had caused inconsis encies in he
de e mina ion o peak a eas), and o he wo ees wi h he same
geno ype in each block, one was andomly chosen o he s udy.
In 2008, when he lea es we e collec ed, he ees we e on a e age
11 m all. The he mal g owing season (i.e., he pe iod when he
mean daily empe a u e emains abo e 5◦C) s a ed on Ap il 27,
ended on Oc obe 29 and had a mean empe a u e o 10.8◦C
(Finnish Me eo ological Ins i u e).
To analyze he g een lea chemis y, a sample o 30 lea es
(e e y second non-damaged lea om he ip o a sou h-side
b anch, g owing a he minimum heigh o 150 cm) was collec ed
om he ees in i e eplica e blocks (n=5) on June 26. The
collec ed lea es we e immedia ely ozen in liquid ni ogen. Fo
collec ing senescen lea es, wo sou h-side b anches (a he heigh
o 140–300 cm) o each ee we e enclosed in mesh bags be o e
lea all (Sep embe 8 o 10) in all six blocks (n=6, he numbe
o eplica es was inc eased o i he numbe o blocks in he si e
o decomposi ion; see below). The mesh bags we e collec ed a e
lea all (Oc obe 28 o 30), hei con en s we e pooled wi hin a
ee and andom subsamples o lea es we e aken o labo a o y
analyses. Remaining lea es we e s o ed in plas ic bags in 4◦C
un il No embe 5, when 10-g (d y mass equi alen ) samples
we e used o es ablish li e pa ches on he g ound o a clea -
cu , B. pendula-Pinus syl es is o es si e in Loppi, sou h Finland
(60◦360N, 24◦240E). The soil in his si e is pos -glacial so ed ine
sand wi h a pH o 5.0 and o al C and N concen a ions o 6 and
0.3%, espec i ely, in he uppe 0–5 cm laye (Mikola e al., 2014).
The g ound laye ege a ion is domina ed by a e n P e idium
aquilinum (L.) Kuhn, g asses Calamag os is a undinacea (L.)
Ro h and Deschampsia lexuosa (L.) T in., and dwa sh ubs
Vaccinium my illus L. and Vaccinium i is-idea L. (Mikola e al.,
2014). Using a o es si e ins ead o he Kuikannii y si e (which
was es ablished on an ag icul u al ield), we ensu ed ha he
li e and me aboli es we e subjec ed o decomposi ion in a o es
en i onmen , whe e he decompose s a e adap ed o ee li e
decomposi ion. The li e pa ches we e alloca ed o six eplica e
blocks ( ollowing he blocking ac o in he Kuikannii y si e) and
we e co e ed, bu no enclosed, wi h a 1-mm mesh o p e en
disappea ance and mixing o lea es. The senescen lea es used
in he pa ches we e no d ied o d y mass measu emen s in
o de o p ese e he mic obes, such as endophy es (Saikkonen
e al., 2003, 2015), g owing on he lea es. The li e pa ches we e
allowed o decompose in he ield un il June 24, 2009 (i.e., o
231 days) when 20 pa ly decomposed lea es we e andomly
selec ed om each pa ch, s o ed in –76◦C and used o seconda y
me aboli e analyses. The mean li e mass loss a his s age o
decomposi ion was 9% (Sil e e al., unpublished da a).
Analyses o Seconda y Me aboli es
The subsamples o lea es and li e ha we e used o ex ac ing
seconda y me aboli es we e g ound in liquid ni ogen and s o ed
in –76◦C un il analysed. Upon analysis, he samples we e d ied
o e nigh in a acuum cen i uge concen a o . Samples o
40 ±5 mg we e hen g ound using a s ainless s eel bead in a
TissueLyse o 5 min, ex ac ed in 1 ml o 80% me hanol o
30 min, cen i uged (13000 pm, 2 min), and again ex ac ed
wi h 1 ml o 100% o 10 min. The supe na an s we e d ied in
a acuum concen a o a 45◦C and s o ed a 4◦C.
Lignin was de e mined om he p ecipi an s (5 o 10 mg o
lea o li e sample, espec i ely) using he me hod desc ibed
in B inkmann e al. (2002) and he weigh o he ob ained
biomass pelle was used as an es ima e o he lignin con en .
The d ied supe na an was hen esuspended in 1.8 ml o 100%
me hanol and he concen a ion o condensed annins (syn.
p oan hocyanidins) was de e mined om a 100-µl aliquo o
me hanol esuspension using he acid bu anol assay (Hage man,
2002). In he assay, 900 µl bu anol-HCl (5%) and 10 µl Fe3+-
eagen we e added and he suspension was incuba ed a 90◦C
o 50 min. A e being cooled wi h ice, he abso bance o he
suspension was measu ed a 550 nm using cyanidin chlo ide
(Ex asyn hese, Genay, F ance) as a quan i ica ion s anda d.
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Fo quan i ying he concen a ions o small-molecula
phenolics and i e penoids, a 500-µl aliquo o he me hanol
esuspension was d ied in a acuum cen i uge a 45◦C and s o ed
a –20◦C. A e s o age, he samples we e dissol ed in 250 µl
100% me hanol and 250 µl dis illed wa e . High-pe o mance
liquid ch oma og aphy-mass spec ome y (HPLC-MS) was
hen pe o med using The mo Finnigan LC wi h he low spli
in o wo be ween a The mo LTQ MS (The mo Finnigan, San
Jose, CA, Uni ed S a es) wi h elec osp ay ioniza ion (ESI) and a
Finnigan PDA de ec o wi h a subsequen Co ona Ul a cha ged
ae osol de ec o . The column was C-18 Luna wi h an inne
diame e o 2 mm, leng h o 150 mm and a pa icle size o 3 µm
(Phenomenex, Denma k). The empe a u e o he ay was se o
18◦C and he column o 40◦C. The sol en s we e (A) 0.1% o mic
acid (Sigma–Ald ich, S einheim, Ge many) in H2O and (B) 0.1 %
o mic acid in ace oni ile (Ch omasol R
g ade, Sigma–Ald ich).
The low was 0.41 ml min−1and he elu ion was pe o med wi h
a g adien as ollows: B s a ed wi h 5%, was inc eased o 50%
by 15 min, o 60% by 35 min, o 85% by 45 min and o 98% by
50 min and hen kep a 98% o 5 min. The column was e u ned
o i s s a ing condi ion wi h 15 min equilib a ion, gi ing a o al
o 70 min o each un. The injec ion olume was 12 µl wi h a
pa ial loop. The MS was un in a posi i e ion mode wi h a mass
ange o 150–1500 m/z. The capilla y empe a u e was kep a
320◦C and he ol age a 5 V. The shea h gas low a e was kep a
20 ml min−1, auxilia y gas low a e a 5 ml min−1and sweep gas
low a e a 5 ml min−1. The ube lens was se o 80 V. The a eas
o he compounds we e in eg a ed using he Xcalibu so wa e.
The compounds we e anno a ed using e en ion imes,
UV spec a and HPLC-MS. Peak picking was done using
Me Align so wa e (Lommen, 2009) based on he g een lea
samples. All analyzed compounds wi h hei e en ion imes
and quan i ica ion ions a e lis ed in Supplemen a y Table S1.
The compounds we e coded in he o de o e en ion ime,
which e lec s inc easing lipophilici y in he e e sed phase LC.
The quan i ica ion was ca ied ou using comme cial s anda ds:
i.e., chlo ogenic acid (Ald ich) o ca eoylquinic acids and
hei de i a i es (CQAs), couma oylquinic acids (CouQAs)
and 3,40-dihyd oxyp opiophenone-3-β-D-glucopy anoside
(DHPPG); (+)-ca echin (Ald ich) o (+)-ca echin; que ce in
3-glucoside (Ex asyn hese) o my ice in, que ce in, and
kaemp e ol de i a i es; and acace in (Ex asyn hese) o
la onoid aglycones. T i e penoids a e epo ed as a bi a y uni s
(peak a ea g−1d y mass). Fo he analyses o he i abili y and
s a is ical signi icance o geno ypic a ia ion in he senescen
lea es and decomposed li e , he small-molecula phenolics
and i e penoids we e g ouped in o in acellula phenolics
(including CQAs, CouQAs, DHPPG, (+)-ca echin, my ice in
glycosides, que ce in glycosides, and kaemp e ol glycosides),
epicu icula la onoid aglycones and epicu icula i e penoids.
In addi ion, he in acellula phenolics we e es ed as subg oups;
i.e., phenolic acids (CQAs and CouQAs), my ice in glycosides,
que ce in glycosides, and kaemp e ol glycosides.
S a is ical Analyses
To a oid a mul i ude o mean es s and o allow easy s a is ical
in e ence in he g aphs (Cumming, 2009), we in e p e ed he
s a is ical signi icance o di e ences be ween g een lea , senescen
lea and decomposing li e me aboli e concen a ions using
85% con idence in e als (CIs) o means. In his app oach,
non-c ossing CIs o wo means deno e a s a is ically signi ican
di e ence be ween he means. I is a common p ac ice o use 95%
CIs, bu hey a e oo conse a i e o es ing mean di e ences
and he bes app oxima ion o α=0.05 is achie ed using 85%
CIs (Pay on e al., 2000). All concen a ion means we e calcula ed
using da a om geno ypes 5, 6, 8, 12, 14, 15, 20, and 25 as hese
we e a ailable o g een lea es.
Since ou plan ma e ial consis ed o a cloned p ogeny
o he selec ed B. pendula geno ypes, all ees wi hin a
geno ype had an equal gene ic s uc u e. In such ma e ial, all
a ia ion ha is ound wi hin geno ypes can be conside ed
o be due o he a ia ion in en i onmen , o due o a
measu emen e o , and all a ia ion ound be ween he
geno ypes o be gene ic (Falcone , 1989). This geno ypic a ia ion
includes bo h he addi i e and non-addi i e componen s,
which canno be sepa a ed in cloned ma e ial, and only
he deg ee o gene ic de e mina ion, i.e., he b oad-sense
he i abili y (H2) can be calcula ed (Falcone , 1989). In ou
s udy, he b oad-sense he i abili ies o he concen a ions o
he h ee me aboli e g oups (in acellula phenolics, epicu icula
la onoid aglycones, and epicu icula i e penoids), in acellula
phenolic subg oups, soluble condensed annins and lignin we e
calcula ed on indi idual plan basis acco ding o he Eq. 1,
whe e σ2
Gand σ2
Ea e a iance componen s o geno ypes and
e o , espec i ely (calcula ed using he SPSS GLM Va iance
componen s p ocedu e). Following common p ac ice in o es
b eeding, he eplica e block was included in he calcula ion
model as a ixed ac o (which emo es he block-scale a ia ion
om e o a iance). This di e s om ou ea lie B. pendula
s udies in a na u al o es si e (Mikola e al., 2014;Sil e e al.,
2015), whe e we we e also in e es ed in he size o he block-scale
en i onmen al a ia ion and ea ed block as a andom ac o .
H2=σ2
G/(σ2
G+σ2
E)(1)
Coe icien s o geno ypic a ia ion (CVG) we e calcula ed
acco ding o he Eq. 2, whe e ¯xis he pheno ypic mean.
CVG=qσ2
G/¯x (2)
The s a is ical signi icance o geno ypic a ia ion in he
concen a ions o he h ee g oups o small-molecula
me aboli es, in acellula phenolic subg oups, condensed
annins, and lignin was es ed using he analysis o a iance.
Following he he i abili y calcula ions, he geno ype was
ea ed as a andom ac o and he ield eplica e block
as a ixed ac o . The homogenei y o esidual a iance
among geno ypes was es ed using he Le ene’s es and he
no mal dis ibu ion o model esiduals using he Shapi o–
Wilk es . To ul ill he a iance and no mali y assump ions,
he da a o condensed annins was log10 ans o med and
he da a o o he me aboli es, excluding lignin, squa e- oo
ans o med. Lignin da a ul illed he assump ions wi hou a
ans o ma ion.
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Paaso e al. Seconda y Chemis y o Bi ch Li e
Rela ions among geno ypes and indi idual seconda y
me aboli es we e u he examined using he p incipal
componen analysis (PCA). Compounds wi h quali a i e
a ia ion we e excluded om hese analyses and he senescen
lea es and decomposed li e we e analyzed sepa a ely as no
all he compounds o senescen lea es we e p esen in li e .
Be o e PCA, bo h columns and ows o he da a ma ix we e
ans o med o ha e a mean o ze o and a s anda d de ia ion o
1. This was done o educe he quan i a i e di e ences among
he compounds and he samples and hus, ocus mo e on
chemical p o iles. Compounds wi h skewed dis ibu ion we e
log10 ans o med. The signi icance o geno ypic a ia ion along
he i s wo PC axes was analyzed using axis sco es and he same
ANOVA models as used o compound concen a ions.
Geno ypic co ela ions among he compound g oups and
be ween he senescen lea es and decomposed li e we e es ed
using he Spea man ank co ela ion analysis. The pe sis ence
o geno ypic a ia ion in he chemical p o iles e ealed by
PCA be ween he senescen lea es and li e was es ed as
ank co ela ions o he geno ype means o PC axis sco es. All
s a is ical analyses we e pe o med using he SPSS 15.0.1 and
SPSS 18 s a is ical packages (SPSS, Chicago, IL, Uni ed S a es)
excep o he PCA, which was pe o med using he SIMCA-P+
so wa e (Ume ics AB, Umeå, Sweden).
RESULTS
Changes in Me aboli e Concen a ions
du ing Lea Senescence and Li e
Decomposi ion
All hose seconda y me aboli es ha we e ound in g een summe
lea es we e also de ec ed in senescen lea es, excep o CQAs
(Figu e 1A). The concen a ions o CouQAs we e on a e age 97%
lowe in he senescen han g een lea es and dec eased below
he de ec ion limi du ing li e decomposi ion (Figu e 1A).
DHPPG concen a ion was 99% lowe in he senescen han g een
lea es and also dec eased below de ec ion du ing decomposi ion
(Figu e 1A).
The dec ease o la onol glycoside concen a ions du ing
lea senescence and li e decomposi ion a ied among he
la onoid subg oups (Figu es 1B–D). Concen a ions o
my ice in glycosides dec eased on a e age by 93% du ing lea
senescence and none o he six compounds was de ec ed in he
decomposed li e (Figu e 1B). In con as , concen a ions o
que ce in and kaemp e ol glycosides dec eased on a e age by 79
and 76% du ing lea senescence and all compounds, excep o
kaemp e ol-3-glucoside, we e also de ec ed in he decomposed
li e (Figu es 1C,D). Based on he compa ison o con idence
in e als, he educ ion in concen a ion du ing lea senescence
was s a is ically signi ican o all la onol glycosides, excep o
kaemp e ol 3-a abino u anoside (Figu es 1B–D). Du ing li e
decomposi ion, he concen a ion o que ce in and kaemp e ol
glycosides dec eased on a e age by 86 and 52%, and excep
o kaemp e ol 3-glucu onide, he dec ease was s a is ically
signi ican in all compounds (Figu es 1C,D).
The concen a ions o epicu icula la onoid aglycones a ied
a lo in he g een lea es, bu displayed ela i ely simila dynamics
du ing lea senescence and li e decomposi ion (Figu e 1E). The
concen a ions we e on a e age 27% lowe in he senescen han
g een lea es and he dec ease was s a is ically signi ican o nine
o he 15 compounds (Figu e 1E). Fo one o he compounds
(F15), he concen a ion inc eased by 70% (Figu e 1E). Du ing
li e decomposi ion, he concen a ions o la onoid aglycones
dec eased on a e age by 51% and he dec ease was s a is ically
signi ican o all compounds (Figu e 1E).
The mean concen a ion o i e penoids dec eased du ing lea
senescence by 25% (Figu e 1F), bu his dec ease was d i en
by one abundan compound T8 ha was anno a ed as 12-
O-ace yl-3-O-malonylbe ula olien iol. When T8 was excluded
om calcula ions, he mean concen a ion o i e penoids
inc eased by 4%, and o he wo oco illol- ype i e penoids,
papy i e ic acid (T7) and i s de i a i e (T6), he inc ease was
s a is ically signi ican (Figu e 1F). Du ing li e decomposi ion,
all i e penoids had pa allel dynamics, he mean concen a ion
dec eased by 55% and he dec ease was s a is ically signi ican o
all compounds (Figu e 1F).
O he polyme s, he concen a ion o lignin inc eased by 51%,
while he concen a ion o condensed annins did no change
du ing lea senescence (Figu e 1G). Du ing li e decomposi ion,
lignin concen a ion inc eased u he by 23%, bu annin
concen a ion dec eased by 87% (Figu e 1G). The (+)-ca echin
concen a ion dec eased by 97% du ing senescence, bu did no
change du ing decomposi ion (Figu e 1G).
Geno ypic Va ia ion in Me aboli e
Concen a ions
The seconda y me aboli es displayed bo h quali a i e (absence
o p esence in only ce ain geno ypes) and quan i a i e ( ound
in all geno ypes, bu in a ying quan i y) geno ypic a ia ion.
Quali a i e a ia ion was ound among la onol glycosides:
he 3-glucu onides we e lacking in ou o he 19 geno ypes
(16, 24, 25 and 30) and he 3-a abino u anosides we e ound
in ou geno ypes only (2, 8, 22, and 23). The quali a i e
a ia ion emained h ough he senescence and decomposi ion
as hese compounds we e also ound in he decomposed li e
(Figu es 1B–D).
Quan i a i e geno ypic a ia ion was ound in all compound
g oups (Figu e 2 and Table 1). O he main me aboli e
g oups, he epicu icula i e penoids (Figu e 2C) had he
highes b oad-sense he i abili y, H2(0.281) and coe icien
o geno ypic a ia ion, CVG(0.138) in he senescen lea es
(Table 1). The o he g oups had e y simila he i abili ies
(0.113–0.121), whe eas he CVG a ied mo e, wi h lignin
and epicu icula la onoid aglycones ha ing lowe CVG han
in acellula phenolics and condensed annins (Table 1). The
geno ypic a ia ion was s a is ically no highly signi ican in
he senescen lea es, excep o i e penoids (Table 1). Among
he in acellula phenolic subg oups, my ice in glycosides and
kaemp e ol glycosides had e y high alues o H2(0.398 and
0.327, espec i ely) and CVG(0.331 and 0.219), while hose o
phenolic acids and que ce in glycosides esembled he alues o
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FIGURE 1 | Mean concen a ions (±85% CI, n= 5 o a abino u anosides and 33–38 o o he compounds, based on geno ypes 5, 6, 8, 12, 14, 15, 20, and 25) o
phenolic compound g oups: (A) phenolic acids and DHPPG, (B) my ice in glycosides, (C) que ce in glycosides, (D) kaemp e ol glycosides, (E) epicu icula la onoid
aglycones, (F) epicu icula i e penoids and (G) he polyme s in Be ula pendula g een lea es, senescen lea es and decomposed lea li e ( he in e al be ween
g een and senescen lea es is 4 mon hs and be ween senescen lea es and decomposed li e 8 mon hs; i e penoids a e epo ed as peak a ea; CQA,
Ca eoylquinic acid; CouQA, Couma oylquinic acid; DHPPG, 3,40-dihyd oxyp opiophenone-3-glucoside; M, My ice in; Q, Que ce in; K, Kaemp e ol; a ,
a abino u anoside; ap, a abinopy anoside; gal, galac oside; glc, glucoside; gl , glucu onide; h, hamnoside).
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FIGURE 2 | Concen a ions o (A) in acellula phenolics, (B) epicu icula la onoid aglycones, (C) epicu icula i e penoids, (D) condensed annins, and (E) lignin
(mean + SE, n= 5–6) in he senescen lea es and decomposed li e o 19 Be ula pendula geno ypes (a anged in a dec easing o de o senescen lea annin
concen a ions).
in acellula phenolics in gene al (Table 1 and Supplemen a y
Figu e S1). Du ing decomposi ion, he H2and CVGinc eased
o in acellula phenolics, epicu icula la onoid aglycones,
and condensed annins, emained he same o epicu icula
i e penoids and dec eased o lignin (Figu e 2 and Table 1). As
a esul , he decomposed li e had s a is ically highly signi ican
geno ypic a ia ion in all compounds excep o lignin ha had
los geno ypic a ia ion du ing decomposi ion (Table 1).
The geno ypic a ia ion in he small-molecula compounds o
senescen lea es was also clea ly isible in he PCA o indi idual
compounds, whe e PC1 ep esen s en i onmen al a ia ion
(P=0.172 o geno ype, P<0.001 o eplica e block) and PC2
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Paaso e al. Seconda y Chemis y o Bi ch Li e
TABLE 1 | The a iance componen s (σ2), b oad-sense he i abili y (H2), pheno ypic mean (¯
x), coe icien o a ia ion (CVG), and Fand Ps a is ics o ANOVA o he
geno ypic a ia ion o seconda y me aboli es in he senescen lea es and decomposed li e o Be ula pendula (G =geno ype, E =e o ; means a e mg g−1d y mass,
excep o epicu icula i e penoids peak a ea g−1d y mass; lignin no ans o med, condensed annins log10- ans o med, o he g oups squa e oo - ans o med; bold
alues deno e s a is ically signi ican geno ype e ec s).
σ2
Gσ2
EH2¯
xCVGF P
Senescen lea es
In acellula phenolics 0.080 0.617 0.115 2.58 0.110 1.69 0.060
Phenolic acids 4.7E-4 0.003 0.120 0.22 0.098 1.61 0.086
My ice in glycosides 0.037 0.056 0.398 0.58 0.331 4.76 <0.001
Que ce in glycosides 0.077 0.539 0.125 2.43 0.114 1.76 0.047
Kaemp e ol glycosides 0.015 0.031 0.327 0.56 0.219 3.58 <0.001
Epicu icula la onoid aglycones 0.005 0.038 0.116 0.92 0.077 1.67 0.062
Epicu icula i e penoids 247 632 0.281 114 0.138 3.08 <0.001
Condensed annins 0.018 0.142 0.113 1.19 0.113 1.95 0.021
Lignin 496 3613 0.121 491 0.045 1.79 0.040
Decomposed li e
In acellula phenolics 0.021 0.057 0.269 1.13 0.128 2.99 <0.001
Que ce in glycosides 0.019 0.058 0.248 1.00 0.139 2.76 0.001
Kaemp e ol glycosides 0.006 0.005 0.528 0.49 0.158 6.96 <0.001
Epicu icula la onoid aglycones 0.005 0.020 0.200 0.66 0.107 2.40 0.004
Epicu icula i e penoids 115 352 0.246 79 0.136 2.75 0.001
Condensed annins 0.011 0.032 0.263 0.47 0.223 3.01 <0.001
Lignin 29 874 0.032 614 0.009 1.19 0.289
mos ly geno ypic a ia ion (P<0.001 o geno ype, P=0.042 o
block) (Figu e 3A). In he decomposed li e , geno ypic a ia ion
was signi ican along bo h he PC1 (P=0.018 o geno ype,
P<0.001 o block) and he PC2 (P<0.001 o geno ype,
P=0.003 o block) (Figu e 3B). The anks o geno ype mean
sco es co ela ed posi i ely be ween he senescen lea es and
decomposed li e o PC2 (ρ=0.87, P<0.001), bu no o
PC1 (ρ=–0.43, P=0.064). The geno ype 16 was mos dis inc
om o he s in bo h senescen lea es and decomposed li e
(Figu es 3A,B). T i e penoids and he mos lipophilic la onoid
aglycones (F11-F15) we e he compounds ha bes explained he
geno ypic a ia ion along he PC axes (Figu es 3C,D). In he
decomposed li e , ou i e penoids, including papy i e ic acid
(T7) and i s de i a i e (T6), o med a igh clus e sepa a ed om
he es o he compounds (Figu e 3D).
Geno ypic Co ela ions among
Me aboli es
The geno ype mean concen a ions o he wo epicu icula
compound g oups – la onoid aglycones and i e penoids – we e
posi i ely co ela ed in he senescen lea es, whe eas he mean
concen a ions o condensed annins co ela ed nega i ely wi h
he concen a ions o la onoid aglycones and lignin (Table 2
and Supplemen a y Figu e S2). The posi i e co ela ion be ween
he la onoid aglycones and i e penoids also emained in he
decomposed li e (Table 2). Among he in acellula phenolic
subg oups o he senescen lea es, concen a ions o phenolic
acids co ela ed posi i ely wi h he concen a ions o que ce in
glycosides (ρ=0.63, P=0.004, n=19) and kaemp e ol
glycosides (ρ=0.52, P=0.023), which also co ela ed wi h
each o he (ρ=0.63, P=0.004). The anks o geno ype mean
concen a ions co ela ed posi i ely be ween he senescen lea es
and decomposed li e o in acellula phenolics (ρ=0.70,
P=0.001, n=19), la onoid aglycones (ρ=0.46, P=0.048),
i e penoids (ρ=0.75, P<0.001) and condensed annins
(ρ=0.62, P=0.004), bu no o lignin (ρ=0.16, P=0.514)
(Figu e 2).
DISCUSSION
Ou esul s show ha al hough he concen a ions o many
seconda y me aboli es dec eased signi ican ly du ing B. pendula
lea senescence, all me aboli es excep o ca eoylquinic acids
(CQAs) emained in he senescen lea es. As we hypo hesized,
he emaining me aboli es also exhibi ed signi ican geno ypic
a ia ion wi h high b oad-sense he i abili ies and coe icien s o
geno ypic a ia ion. Du ing decomposi ion, mos me aboli es
dec eased in concen a ion, sugges ing ha hey we e
decomposed as e han he li e ma e ial on a e age, bu
he geno ypic a ia ion was pe sis en . This was mani es ed
by he inc easing he i abili ies and coe icien s o geno ypic
a ia ion o he in acellula phenolics, su ace la onoid
aglycones and condensed annins du ing li e decomposi ion.
Con i ming he pe sis ence o geno ypic a ia ion, he geno ype
anks in me aboli e concen a ions emained s able unde
ield condi ions and mic obial deg ada ion. Conside ing ha
seconda y me aboli es can a ec li e decomposi ion and
nu ien cycling (Hä enschwile and Vi ousek, 2000;Schwei ze
e al., 2004), hese esul s sugges ha by ac ing on he olia
seconda y me aboli e p o iles o B. pendula popula ions, selec ion
can be a signi ican d i e o li e decomposi ion. Lignin was
an impo an excep ion among he compounds, howe e , as
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FIGURE 3 | P incipal componen analysis (PCA) g aphs o he seconda y me aboli e da a in (A) senescen lea es and (B) decomposed li e and he loadings o he
compounds esponsible o he a ia ion on he PC axes as p(co ) alues in (C) senescen lea es and (D) decomposed li e . In (A,B), he 19 geno ypes a e shown
as mean axes sco es wi h he e ical and ho izon al e o ba s depic ing ±1 SE (n= 4–6). CouQAs, Couma oylquinic acids; F, Fla onoid aglycone; FG, Fla onol
glycosides; Kgal, Kaemp e ol 3-galac oside; K h, Kaemp e ol 3- hamnoside; Mgal, My ice in 3-galac oside; Mglc, My ice in 3-glucoside; Qap, Que ce in
3-a abinopy anoside; Qgal, Que ce in 3-galac oside; Q h, Que ce in 3- hamnoside; T, T i e penoid.
TABLE 2 | Rank co ela ion coe icien s (Spea man’s ho) o geno ype mean concen a ions o seconda y me aboli es in he senescen lea es and decomposed li e o
Be ula pendula (n=19; ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001; bold alues deno e s a is ically signi ican co ela ions).
Epicu icula la onoid aglycones Epicu icula i e penoids Condensed annins Lignin
Senescen lea es
In acellula phenolics 0.05 0.14 0.17 <0.01
Epicu icula la onoid aglycones 0.75∗∗∗ −0.47∗0.25
Epicu icula i e penoids −0.43 0.15
Condensed annins −0.51∗
Decomposed li e
In acellula phenolics −0.02 −0.05 0.15 −0.37
Epicu icula la onoid aglycones 0.71∗∗ 0.29 −0.13
Epicu icula i e penoids 0.20 −0.25
Condensed annins −0.37
lignin concen a ions inc eased du ing lea senescence and
li e decomposi ion and he geno ypic a ia ion disappea ed
du ing decomposi ion. Mo eo e , ou esul s show ha he
geno ype means o seconda y me aboli e concen a ions, like
hose o condensed annins and lignin, can be nega i ely
co ela ed in he senescen lea es. As lignin (Melillo e al., 1982;
Hobbie e al., 2006;Talbo and T esede , 2012) and condensed
annins (Schwei ze e al., 2004, 2008a) can bo h es ic li e
decomposi ion, an associa ion be ween li e decomposi ion
a e and a concen a ion g adien o one compound could
be canceled ou by an in e se g adien o he o he . In such
case, selec ion ac ing on he concen a ion o ei he compound
in he g een lea es migh no lead o a signi ican change
in li e decomposi ion a e in he popula ion. Also, equally
impo an is o ecognize he ole o o he cha ac e is ics o li e
chemis y, such as concen a ions o ni ogen (Sil e e al., 2007)
and mic onu ien s (Makkonen e al., 2012; Ga cía-Palacios
e al., 2016a), when weighing he oppo uni y o selec ion o
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